The goals of this project are to characterize the mechanisms by which ploidy is limited in Saccharomyces cerevisiae. Yeast with ploidies as high as 6n have been easily generated, however, it has not been possible using low-resolution techniques to generate viable yeast with a ploidy above 6n (greater than 6n). Limitations in polyploidy are also seen in other eukarytoic cells in a cell-type specific manner. Many liver hepatocytes are 4n, but, the number that achieve a ploidy of 8n are limited. In addition, the proliferative activity of rat liver carcinoma cells decreases in an inverse proportion to their ploidy. In contrast, megakaryocytes can reach as high as 64n. The specific aims of this research proposal are the following: (1) Accurately identify the limits on increasing polyploidy in yeast. Complementary markers will be used to select for viable 8n polyploids from crosses using large numbers of parental cells. The frequency with which 8n polyploids arise (if 8n polyploidy is possible in these strains) will be determined. The phenotypes of isolated 8n yeast will be carefully examined. (2) Identify genes that are involved in limiting polyploidy. Seventeen genes are known to have ploidy-regulated expression. The role that the altered expression of each may play in the prevention of viable greater than 6n strains will be analyzed by overexpressing those that are repressed in polyploids and disrupting those that are induced. (3) Identify differences in the patterns of genes expressed during conjugation when comparing n x n and polyploid crosses. Whole genome mRNA expression microarrays will be used to analyze yeast in n x n, 2n x 2n, and 4n x 4n crosses. Genes that are differentially expressed between these crosses will be identified and examined further.